The objective of this research is to use a combination of EPR, ENDOR, NMR, X-ray crystallographic, synthetic and computational methods to investigate the structure, function, and inhibition of isoprenoid biosynthesis enzymes of interest as drug targets for treating, primarily, tropical diseases. In Aim 1, we will investigate GcpE, an enzyme involved in isoprenoid biosynthesis in malaria parasites. We hypothesize that its mechanism of action involves unusual metallacycles and that similar metallacycles form with novel alkyne inhibitors. GcpE is an excellent target for the development of novel anti-infectives since it is essential for pathogen survival, is not found in humans, and we have now identified novel inhibitors. In Aim 2 we will carry out a similar series of investigations of the following enzyme (from P. falciparum) in the pathway, LytB, and we hypothesize that because of similarities in their mechanism of action, LytB inhibitors will also inhibit GcpE leading, in cells, to synergistic activity. The third Aim is to develop novel anti-malarials that function by blocking carotenoid and quinone biosynthesis. Carotenoids act to remove reactive oxygen species (ROS, from hemoglobin catabolism) and we hypothesize that carotenoid (and quinone) biosynthesis inhibitors will synergize with current anti-malarials (that enhance ROS formation), reducing drug resistance. The final Aim involves investigation of three other high-value targets: in trypanosomatid parasites, farnesyl diphosphate synthase and hexokinase, in bacteria, undecaprenyl diphosphate synthase. All are essential for survival and sub-micromolar leads have already been identified by us. If successful, the work will provide many new insights into enzyme mechanisms, as well as new drug leads for many global infectious diseases. PUBLIC HEALTH RELEVANCE: This project is aimed at developing new leads for treating infectious diseases, primarily malaria. Focus will be on developing inhibitors for three unique targets in malaria parasites, and on the use of novel drugs against sleeping sickness.